Biotechnol Lett (2012) 34:20492053 DOI 10.

1007/s10529-012-0996-2

ORIGINAL RESEARCH PAPER

Proling of carotenoids in six microalgae (Eustigmatophyceae) and assessment of their b-carotene productions in bubble column photobioreactor
Zhen Li Mingzhe Sun Qiyu Li Aifen Li Chengwu Zhang

Received: 9 April 2012 / Accepted: 22 June 2012 / Published online: 11 July 2012 Springer Science+Business Media B.V. 2012

Abstract The proles of carotenoids and production of b-carotene by six eustigmatophytes, Eustigmatos magnus, Eustigmatos polyphem, Eustigmatos vischeri, Vischeria helvetica, Vischeria punctata and Vischeria stellata, grown in a bubble column photobioreactor were measured. All eustigmatophytes contained bcarotene, violaxanthin and vaucheriaxanthin as their major carotenoids and accumulated large amount of bcarotene, which accounted for over 50 % of total carotenoids. Maximum intracellular b-carotene contents ranged 1.53.5 % of dry wt and in V. stellata it reached 5.9 % dry wt, accompanied by a biomass dry wt [7.3 g/l, with the highest up to 9.8 g/l. These eustigmatophytes are thus promising producers of bcarotene. Keywords b-Carotene Carotenoid Eustigmatophyceae Microalgae Photobioreactor

Introduction Microalgae are good sources of commercially valuable carotenoids, such as b-carotene. b-Carotene can be used as a scavenger of free radicals (Burton and Ingold 1984), activator of gene expression (Stahl and Sies 1997) and stimulant of the immune response (Chew and Park 2004). Carotenoids have received particular attention owing to their increasing demand and wide applications in food, aquaculture, cosmetic and pharmaceutical industries as colorants, feed additives, antioxidants, anti-tumor agents and heartdisease prevention agents (Prieto et al. 2011). However, microalgae with high potentials of producing valuable carotenoids remain limited to several microalgal genera up to date, e.g., b-carotene in Dunaliella and astaxanthin in Haematococcus. Therefore, exploiting novel microalgal sources of valuable carotenoids is of growing interest. Eustigmatophyceae is a small microalgal class with a few unicellular coccoid microalgae classied into a small number of genera. Microalgae of this class are characterized by the presence of only chlorophyll a and violaxanthin, vaucheriaxanthin and b-carotene as the major carotenoids (Whittle 1976). Nannochloropsis spp. (Eustigmatophyceae) were reported by n et al. (2000) as novel microalgal sources of Lubia commercially valuable carotenoids (astaxanthin and canthaxanthin). But, it is not clear whether other genera (e.g., Eustigmatos and Vischeria) of this class have similar commercial potentials. Little information

Zhen Li and Mingzhe Sun contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s10529-012-0996-2) contains supplementary material, which is available to authorized users.
Z. Li M. Sun Q. Li A. Li (&) C. Zhang Institute of Hydrobiology, School of Life Science and Technology, Jinan University, Huangpu Avenue No. 601, Tianhe District, Guangzhou 510632, China e-mail: tiger@jnu.edu.cn

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2050 Fig. 1 The schematic diagram a of bubble column photobioreactor and b photograph of biomass (V. stellata) harvested. The photobioreactor was made of thin glass with a 3 cm diameter (optical-path) and a length of 60 cm

Biotechnol Lett (2012) 34:20492053

A
Rubber plug

Glass inlet Outlet

Light source

60 cm

3 cm

is available on the specic carotenoid proles of these genera. Furthermore, we had observed the Eustigmatos and Vischeria microalgae changed color with aging, particularly in the late stationary stage of growth when their cultures became yellow-orange, which was probably related to intracellular pigmentation of b-carotene and a decreased content of chlorophylls. This was similar to Dunaliella as indicated by Ben-Amotz and Avron (1983). With the above considerations, the aims of this paper were to prole the carotenoids of six eustigmatophytes belonging to two genera (Eustigmatos and Vischeria) and to evaluate their potentials of b-carotene production in a bubble column photobioreactor.

and NaNO3 (1.5 g/l). They were grown at pH 7.5 and at 23 C in a bubble column photobioreactor (60 9 3 cm) with a working volume of approx. 300 ml (Fig. 1a). Cells were initially grown in a bubble column bioreactor (60 9 6 cm) for 6 days and then inoculated into the nal bioreactor to give an OD750 of 0.7. Bioreactors were aerated with compressed air containing 1 % CO2 (v/v) at approx. 2 l/min and irradiated continuously from one side with daylight uorescence lamps giving 300 lmol photons m-2 s-1. Measurement of cell growth and dry weight Growth was monitored daily from the OD750 and by counting cell numbers. For biomass (dry weight) determination, triplicate culture (510 ml) was diluted with distilled water and then ltered through preweighed 0.45 lm membrane lters. Filtered cells were then dried to a constant weight at 80 C. Cells were also harvested by centrifugation (5,0009g for 5 min), freeze-dried and stored at -20 C for later analysis. Analysis of carotenoids Carotenoids were extracted from freeze-dried samples with methanol containing 0.1 % (w/v) butylated hydroxytoluene, using initially ultrasonics and then a magnetic stirrer. After centrifugation, the supernatant was collected and the pellet was re-extracted until it was colorless. The combined supernatants were ltered through a 0.45 lm nylon membrane lter and analyzed immediately by HPLC. All procedures

Materials and methods Microorganisms and culture conditions Six eustigmatophytes, Eustigmatos magnus SAG36.89, E. polyphem SAG38.84, E. vischeri SAG860-1, Vischeria helvetica SAG876-1, V. punctata SAG887-1 and V. stellata SAG33.83) were from the Institute of Hydrobiology, Jinan University (China). They were maintained in BG-11 medium (Allen and Stainer 1968) with the following composition: K2HPO43H2O (0.04 g/l), MgSO47H2O (0.075 g/l), CaCl22H2O (0.036 g/l), FeCl36H2O (0.006 g/l), Na2CO3 (0.02 g/l), H3BO3 (2.86 g/l), MnCl24H2O (1.81 g/l), Na2MoO42H2O (0.391 g/l), CuSO45H2O (0.079 g/l), ZnSO47H2O (0.22 g/l), EDTA (0.001 g/l), Citric acid (0.006 g/l)

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were conducted under dim light to prevent carotenoid photo-oxidation. Carotenoids were analyzed by HPLC using a C18 reversed phase column (5 lm, 250 9 4.6 mm) coupled with a C18 guard column. The binary mobile phase, modied from the method of Wright et al. (1991), was acetonitrile/water (9:1, v/v) (eluent A) and 100 % ethyl acetate (eluent B). Carotenoids were eluted at 1 ml/min as follows: initial 100 % A and 0 % B, 020 min linear gradient to 100 % B, 2022 min 100 % B, 2223 min return to 100 % A, 2325 min 100 % A for re-equilibration (Li et al. 2012). Carotenoids were identied by comparing retention time and visible absorption spectra with authentic standards. The identication of additional peaks was carried out by comparing their spectral data with reported values (Wright et al. 1991). Quantication of carotenoids was carried out using external calibration curves and those with authentic standards unavailable, were quantied by comparison of their peak areas with that of violaxanthin.

helvetica, V. punctata and V. stellata. b-Carotene was the predominant carotenoid in each microalga, accounting for over 50 % of total carotenoids. How n et al. (2000), violaxanthin ever, according to Lubia rather than b-carotene accounted for up to 60 % of total carotenoids in eustigmatophycean microalgae. We believe that this difference is probably due to different growth stage in which these eustigmatophytes were analyzed and with different culture conditions. Also, carotenoid proles might differ between species. The concentrations of b-carotene in each microalga, particularly in the three species of Vischeria, were high at over 25 mg/g. V. stellata was the most attractive, which accumulated up to 54.5 mg carotenoids/g with b-carotene accounting for 61.5 %. The chlorophyll a contents of all the microalgae were relatively low at approx. 24 mg/g. b-Carotene production by the six eustigmatophytes in bubble column photobioreactor Biomass kinetics of the six eustigmatophytes grown in a photobioreactor are shown in Fig. 2. Each microalga grew quickly and achieved biomasses exceeding 9 g/l after 18 days, with the highest one reaching 9.8 g/l with E. polyphem. Generally, all eustigmatophytes accumulated carotenoids with ageing (Fig. 3a). After 18 days, V. stellata

Results Carotenoid proles of six eustigmatophytes Table 1 shows the carotenoid proles of six eustigmatophytes, E. magnus, E. polyphem, E. vischeri, V.

Table 1 Carotenoids proles of the six eustigmatophycean microalgae Carotenoids % of total carotenoids E. magnus Violaxanthin Vaucheriaxanthin Zeaxanthin Luteoxanthin Antheraxanthin b-Carotene Others
c

E. polyphem 2.5 12.7 3.2 4.9 2.2 50.5 22 14.3 1.1 2.2 0.3

E. vischeri 10.0 14.1 1.9 3.5 2.6 53 14.9 19.4 1.6 2.5 0.2

V. helvetica 12.1 10.5 1.2 4.2 2.3 58.4 11.3 25.4 2.0 3.9 0.6

V. punctata 11.3 11.8 1.7 4.7 2.4 56.7 11.4 33.2 1.5 1.9 0.5

V. stellata 13.3 7.6 1.6 3.6 1.8 61.5 10.6 54.5 2.3 2.1 0.7

14.6a 12.0 2.6 2.1 1.9 52.8 14.1 25.2 1.7b 4.3 0.4b

Total carotenoids (mg/g cell dry wt) Chlorophyll a (mg/g cell dry wt)

The microalgae used for this part of study were cultivated in the Erlenmeyer asks under the irradiance of approx. 150 lmol photons m-2 s-1 and harvested on day 18
a b c

Mean of three trials Mean SD (n = 3) Other carotenoids = 90 -cis-neoxanthin ? lutein ? vaucheriaxanthin-ester ? neoxanthin-esteried ? the unidentied

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2052 Fig. 2 Biomass kinetics of the six eustigmatophytes of a Eustigmatos and b Vischeria cultivated in a bubble column photobioreactor irradiated at 300 lmol photons m-2 s-1. Each value is expressed as mean SD of three trials

Biotechnol Lett (2012) 34:20492053

A 10
8

B 10
8

Biomass (g/l)

6 4 E. magnus 2 0 0 3 6 9 12 15 18 E. polyphem E. vischeri

Biomass (g/l)

6 4 V. helvetica 2 0 0 3 6 9 12 15 18 V. punctata V. stellata

Time (days)

Time (days)

-Carotene (mg/g dry wt)

achieved the highest carotenoids content reaching 77 mg/g, followed by V. punctata at [48 mg/g. The increase of carotenoids with time was due to the accumulation of b-carotene, though other carotenoids declined simultaneously (data not given). As shown in Fig. 3b, all microalgae, especially the three species of Vischeria, showed large accumulations of b-carotene. Vischeria stellata was the most attractive, achieving bcarotene up to 5.9 % dry wt after 18 days. Due to the large amount of b-carotene, the dried algal powder harvested on day 18 was yellow-orange (Fig. 1b). The percentage of b-carotene in the total carotenoids increased with time (Fig. 3), accounting for approx. 70 % after 18 days. This would then make its recovery and purication relatively easy.

A
Total carotenoids (mg/g dry wt)
90 80 70 60 50 40 30 20 10 0 Day 0 Day 6 Day 12 Day 18

90 80 70 60 50 40 30 20 10 0

Discussion The six eustigmatophytes studied in this paper are rarely studied for their carotenoids and biomasses. n et al. (2000) studied several eustigmatophytes Lubia (Nannochloropsis spp.) and found they accumulated high biomasses and high contents of carotenoids (canthaxanthin and astaxanthin), which accounted for 0.7 % of cell dry wt. However, their intracellular bcarotene were all at a barely detectable concentration. In contrast, the six eustigmatophytes of Eustigmatos and Vischeria studied here were rst found to accumulate large amount of b-carotene. To date, microalgae which accumulate large amounts of b-carotene are limited to species of Dunaliella, which can accumulate it up to 10 % dry wt under stress conditions (Ben-Amotz 1999). The intracellular b-carotene accumulated by the six

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Fig. 3 Time course of total carotenoids content a and intracellular b-carotene accumulation b of the 6 eustigmatophytes cultivated in a bubble column photobioreactor irradiated at 300 lmol photons m-2 s-1. The values are means of three replicates SD

eustigmatophytes examined here was not as much as in Dunaliella but it is probable that their b-carotene contents can be increased by improving the culture

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2053 Ben-Amotz A (1995) New mode of Dunaliella biotechnology: two-phase growth for b-carotene production. J Appl Phycol 7:6568 Ben-Amotz A (1999) Dunaliella b-carotene: from science to commerce. In: Seckbach J (ed) Enigmatic microorganisms and life in extreme environments. Kluwer Academic Publisher, The Netherlands, pp 401410 Ben-Amotz A, Avron M (1983) On the factors which determine massive b-carotene accumulation in the halotolerant alga Dunaliella bardawil. Plant Physiol 72:593597 Burton GW, Ingold KU (1984) Beta-carotene: an unusual type of lipid antioxidant. Science 224:569573 Chew BP, Park JS (2004) Carotenoid action on the immune response. J Nutr 134:257S261S a-Gonza lez M, Moreno J, Manzano JC, Florencio FJ, Garc Guerrero MG (2005) Production of Dunaliella salina biomass rich in 9-cis-b-carotene and lutein in a closed tubular photobioreactor. J Biochem 115:8190 mez P, Barriga A, Cifuentes AS, Gonza lez M (2003) Effect Go of salinity on the quantity and quality of carotenoids accumulated by Dunaliella salina (strain CONC-007) and Dunaliella bardawil (strain ATCC30861) chlorophyta. Biol Res 36:185192 Li Z, Ma XQ, Li AF, Zhang CW (2012) A novel potential source of b-carotene: Eustigmatos cf. polyphem (Eustigmatophyceae) and pilot b-carotene production in bubble column and at panel photobioreactors. Bioresour Technol 117: 257263 n LM, Montero O, Moreno-Garrido I et al (2000) NanLubia nochloropsis (Eustigmatophyceae) as source of commercially valuable pigments. J Appl Phycol 12:249255 avate JP, Garc a-Gonza lez M (2011) Prieto A, Pedro Can Assessment of carotenoid production by Dunaliella salina in different culture systems and operation regimes. J Biotechnol 151:180185 Stahl W, Sies H (1997) Carotenoids and intercellular communication via gap junctions. Int J Vitam Nutr Res 67: 364367 Whittle SJ (1976) The major chloroplast pigments of Chlorobotrys regularis (West) Bohlin (Eustigmatophyceae) and Ophiocytium majus Naegeli (Xanthophyceae). Br Phycol J 11:111114

conditions. Further, the six eustigmatophytes accumulated higher biomasses, compared with Dunaliella species, which generally yield a biomass less than 2 g/ mez et al. 2003; Garc al (Ben-Amotz 1995; Go Gonzalez et al. 2005). From this point of view, the six eustigmatophytes appeared to be comparable with or more productive than them, though their intracellular b-carotene contents are higher. The reason why these eustigmatophytes accumulate large amount of b-carotene is not clear at present. But, since b-carotene has diverse physiological functions as described in the Introduction, it is possible that these eustigmatophytes synthesize more b-carotene to protect themselves from the damage caused by ageing or environmental stresses, such as nutrient depletion and high light irradiance. This is similar to the explanation given by Ben-Amotz and Avron (1983) for the massive b-carotene accumulation in Dunaliella species.

Conclusions All six rarely characterized eustigmatophytes contained b-carotene, violaxanthin and vaucheriaxanthin as the major carotenoids. Growth of them in a bubble column photobioreactor showed that they could produce large amount of b-carotene. Hence, we believe these eustigmatophytes, particularly V. stellata, are the potential natural producers of b-carotene.
Acknowledgments This project was supported by the National Natural Science Foundation of China (Grant No. 21311057 and No. 31170337).

References
Allen MM, Stainer RY (1968) Growth and division of some unicellular blue-green algae. J Gen Microbiol 51:199202

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